Synthesis and some pharmacological properties of [1-. beta.-mercapto

Chem. , 1975, 18 (12), pp 1262–1264. DOI: 10.1021/jm00246a021. Publication Date: December 1975. ACS Legacy Archive. Cite this:J. Med. Chem. 18, 12 ...
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Journal of Medicinal Chemistry, 1975, Vol. 18, No. 12

Notes

Synthesis and Some Pharmacological Properties of [ 1-P-Mercapto-&&diethylpropionic acid,2-(3,5-dibromo-~-tyrosine)]oxytocin+ Edwin 0. Lundell,* Clark W. Smith, and Martha F. Ferger Department of Chemistry, Cornell University, Ithaca, New York 14853. Received M a y 25, 1975 The synthesis of the protected polypeptide precursor of [ l-P-mercapto-P,P-diethylpropionic acid,2-(3,5-dibromo-~tyrosine)]oxytocin was performed in a stepwise manner by solution techniques. This analog of oxytocin has two modifications, each of which taken alone gives analogs which inhibit some of the pharmacological responses to oxytocin. The S-ethylcarbamoyl protecting groups of P-Mpa(B-Etz)(Ec)-Dbt-Ile-Gln-Asn-Cys(Ec)-Pro~Leu-Gly-NHz were removed in refluxing liquid "3, and the resulting disulfhydryl compound was oxidatively cyclized in H20-MeOH with ICH2CHzI. Purification was effected by partition chromatography and gel filtration. The analog possesses antioxytocic (pA2 = 7.08) and antiavian vasodepressor (pAz = 7.38) activities but has neither agonist nor antagonist activity in the rat pressor assay. These potencies are close to those exhibited by [ 1-P-mercaptopropionic acid,2-(3,5-dibromo-L-tyrosine)]oxytocinbut different from those of [ 1-P-mercapto-P,P-diethylpropionic acidloxytocin.

The introduction of two alkyl substituents on the @ carbon a t position 1of oxytocin 1

2

3

4

5

6

7

8

9

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-GI y-NH, .

i

or deamino-oxytocin2 ( [ 1-/3-mercaptopropionic acidloxytocin) has been shown to generate analogs which are potent competitive inhibitors of the oxytocic and avian vasodepressor (AVD) activities of o ~ y t o c i n . ~Three -~ of these acidloxytoanalogs ([ l-p-mercapto-/3,P-dimethylpropionic acidloxytocin, and cin, [1-@-mercapto-p,p-diethylpropionic [l-@-mercapto-/3,@-pentamethylenepropionic acidloxytocin) have also been shown to be weak inhibitors of the rat In addition, several pressor effect of 8-lysine-va~opressin.~~~ analogs of oxytocin with modifications at position 2 have been prepared which have inhibitory properties. Among these are analogs with p-alkoxyphenylalanine or p-alkylphenylalanine residues in this position' and analogs in which one or more of the hydrogen atoms of the aromatic ring of the tyrosine residue has been formally replaced by iodines or bromine.9 These halogen-containing analogs, [2o-iodotyrosine]oxytocinand [ 1-(3-mercaptopropionic acid,2(3,5-dibromotyrosine)]oxytocin,are potent inhibitors with antioxytocic potencies approaching that of [ 1-P-mercaptop,p-diethylpropionic acidloxytocin, which is one of the most potent reversible inhibitors studied in this laboratory. In order to determine the effect of incorporating into one analog two of these potent inhibitory modifications, the synthesis of [l-p-mercapto-p,P-diethylpropionicacid,2(3,5-dibromotyrosine)]oxytocin ( [P-Mpa(P-Etz) ,Dbt2]oxytocin) was undertaken. The required intermediate, p-

AVD responses to synthetic oxytocin in the standard assay systems (see Experimental Section). Its inhibitory potencies in those assays are close to those of [P-Mpa1,Dbt2]oxytocin but significantly ( p < 0.01)less than those of [&Mpa(PEtz)l]oxytocin. In the rat pressor assay, no agonist or antagonist activity was observed for [P-Mpa(P-Etz)l,Dbt2]oxytocin in doses up to 18 pgl100 g of body weight. [pMpa1,Dbt2]oxytocinalso lacks agonist or antagonist activity in the pressor assay, while [@-Mpa(fi-Etz)l]oxytocin possesses a weak antagonist activity. Thus in this study, the inhibitory potencies of the disubstituted analog are limited to the potencies of the less potent analog containing only one inhibitory modification, [fl-Mpa1,Dbt2]oxytocin.

Experimental Section

All melting points were determined in open capillary tubes and are corrected. Thin-layer chromatography was performed on precoated glass plates of silica gel GF 254 (0.25 mm, E. Merck) in the following solvent systems: (A) CHC13-MeOH-AcOH (3:l:l); (B) BuOH-ACOH-H~O (3:l:l); (C) BuOH-pyridine-AcOH-HZO (15: 10:3:12); (D) BuOH-pyridine-HzO (20:lO:ll); (E) acetone-AcOHH20 (8:l:l). The load size was 10-30 fig, and chromatogram lengths were 100-150 mm. Detection was made by chlorination followed by NaI-starch treatment. In all cases, unless otherwise noted, single symmetrical spots were observed for purified material. Progress of the condensation reactions was followed by the quantitative ninhydrin tesL6J6 Amino acid analysis was performed on a Beckman Model 116 amino acid analyzer using a single column ~ y s t e m . ~ Optical .'~ rotations were determined on a PerkinElmer Model 141 polarimeter. Where analyses are indicated only by the symbols of the elements, analytical results obtained for the elements were within f0.4% of the theoretical values. Where compounds are formulated as containing solvent, this does not necessarily imply that they are defined solvates; particularly in the case of amorphous products, solvent retention may be due to mild Mpa(P-Et2)(Ec)-Dbt-Ile-Gln-Asn-Cys(Ec)-Pro-Leu-Glydrying conditions (24 hr at 22'/0.005 Torr over Pzos). "2, was prepared by the stepwise elongation of H-IleBoc-Dbt-Ile-Gln-Am-Cye(Ec)-Pro-Leu-Gly-NHz (1). 2-IleGln-Asn-Cys(Ec)-Pro-Leu-Gly-NH2,lo using Boc-DbtGln-Asn-Cys(E~)-Pro-Leu-Gly-NH2~~ (474 mg, 0.50 mmol) was OPhC131* followed by p-( S-ethylcarbamoy1mercapto)-P,P- treated for 1.5 hr at room temperature with 3.3 N HBr-AcOH (8 ml). The peptide salt was precipitated by the addition of Et20 (30 diethylpropionic acid [P-Mpa(P-Etz)(Ec)]with dicyclohexml), isolated and washed three times with Et20 by centrifugation, ylcarbodiimide (DCC) and 1-hydroxybenzotriazole and dried in vacuo. The residue was dissolved in dimethylformam(HBt).12The S-ethylcarbamoyl groups were removed in reide (DMF) (6 ml) and neutralized to pH 7.5 (Fisher Indicator Soluand the resulting disulfhydryl peptide fluxing liquid "3, tion) with diisopropylethylamine (i-Pr2NEt). Boc-Dbt-OPhCls" was oxidized to the cyclic disulfide with dii0d0ethane.l~ (340 mg, 0.55 mmol) was added. After 24 hr the pH was adjusted The analog was purified by partition c h r ~ m a t o g r a p h y ' ~ back to 7.5 with i-Pr2NEt. After 48 hr the product was precipitated by the addition of EtOAc (30 ml), isolated, washed by centrifuand gel filtration.15 gation with EtOAc and Et20, and dried in vacuo: 525 mg (85%). The highly purified [P-Mpa(/3-Et2)',Dbt2]oxytocinwas The crude product was reprecipitated from DMF with HzO, isolatfound to produce inhibition (Table I) of the oxytocic and ed, washed by centrifugation with H20, EtOH, and EtzO, and dried in vacuo: 436 mg (83%recovery); mp 252-255': [ol2OD-32.9' +The symbols M-Mpa(fi-Et2) and Dbt are used to indicate 8-mercapto(c 0.98, DMF); TLC (A) 0.73 with two trace impurities (0.84 and ;f,/j-diethylpropionic acid and 3,li-dibromo-L-tyrosine, respectively. All other 0.57). symbols follow the recommendations (1971) of the IUPAC-IUB CommisDicyclohexylammonium &(&Ethylcarbamoylmercapto)sion iin Biochemical Nomenclature.' All optically active amino acids are o f &?-diethylpropionate (2). b-(S-Benzylmercapto-@,@-diethylpropthe I , configuration. ionic acid4 (1.00 g, 4.00 mmol) was dissolved in liquid NH3 (100 ml, 'Address correspondence to this author a t Union Carbide Corp., Round freshly distilled from Na). Na (200 mg, 8.7 mmol) was added and Hrook, N..J. 0880.5.

Journal of Medicinal Chemistry, 1975, Vol. 18, No. 12

Notes

1263

Table I. Inhibitory Properties of Certain Oxytocin Analogs Ant iavian Antioxyt ocic Analog

x108

[p-Mpa(p-Et,)' ,Dbt2]oxytocin [P-Mpa' ,Dbt2]oxytocin

[p- Mpa(F- Et,)' ]oxytocin -

2

8.2 (191, u = 1.8 8.9 u = 0.2 5.8 (91,' u = 0.12

PA," 7 -08

7.05b 7.24'

vasodepressor XlO*

z

4.2 (18), u = 2.4 3.6 (16),b u = 1.0 0.78 (81,' u = 0.17 -

PA2

h t i p r e ssor xi07

Z

7.38

No activity

7.44

No activityb

8.11'

5.7 (20),d u = 2.7

PA2

6 .24d

apAz values [H. 0. Schild, Br. J. Pharrnacol., 2, 189 (1947)]represent the negative log to the base 10 of the average molar concentration of an antagonist which will reduce the response to 2x units of pharmacologically active compound (agonist) to the response to x units of agonist. The number of individual determinations is given in parentheses and u is the standard deviation. Specific details of these assays are described by Vavrek et al.4 and by Dyckes et aL6 Synthetic oxytocin was the agonist used in the antioxytocic and antiavian vasodepressor assays and synthetic LVP in the antipressor assays. Concentrations of antagonists were calculated on the basis of a 10-ml tissue bath (oxytocic) or assumed blood volumes of 150 ml for the chicken and 6.7 m1/100 g in the rats. bLundell and Ferger.9 Tavrek et aL4 dDyckeset a1.6

(m)

by rotary evaporation. The residue was lyophilized from AcOH. The lyophilizate was dissolved in 5 ml of the upper phase and 0.5 (1:1:2 aqueml of the lower phase of the system BuOH-C~H~-HZO ous phase, 1.5%pyridine, and 3.5%AcOH) and subjected to partition chr~matography'~ on a 2.2 X 53 cm column of Sephadex G-25 (100-200 mesh) which had been equilibrated with both phases of the solvent system. The product was eluted with upper phase at a flow rate of 11 m l h r and collected in fractions of 1.9 ml. Peptide material was detected by the Folin-Lowry m e t h ~ d . The ' ~ product emerged a t the void volume of the column as a single tailing peak. TLC (B) showed that the product composition was the same across the entire peak area and consisted of a major component at Rf 0.51 with a faint leading impurity. The fractions comprising the peak were pooled, the solvents were removed by rotary evaporation, and the residue was lyophilized from AcOH: 41.0 mg (71%). This mate8-Mpa(8-Etz)(Ec)-Dbt-Ile-Gln-Asn-Cys(Ec)-Pro-Leu-Gly-rial was dissolved in 2 ml of 50% aqueous AcOH and further puriNH2 (3). The Boc group of 1 (296 mg, 0.24 mmol) was removed by fied by gel filtration on a 2.82 X 67 cm column of Sephadex G-25 (200-270 mesh) equilibrated with 20% AcOH. The product was treatment with trifluoroacetic acid (TFA) (2 ml) for 30 min at eluted from the column with 20% AcOH at a flow rate of 27 m l h r room temperature. The peptide salt was precipitated by the addition of EtzO, and the precipitate was isolated, washed by centrifuand collected in fractions of 3.6 ml. Peptide material was detected by reading the absorbency of the eluate at 280 nm. The product gation with EtzO, and dried in vacuo. An EtOH-EtOAc solution of 2 (150 mg, 0.36 mmol) was treated with excess HCl-EtOAc (4.5-5 emerged as a sharp symmetrical peak with a maximum at 70% of the column volume preceded by and resolved from a small peak at N). The precipitated DCHA.HC1 was removed by filtration, and 56% of the column volume. The fractions comprising the major the solvents were removed from the filtrate by rotary evaporation leaving a colorless oil. The oil was dissolved in 1.00 ml of DMF and peak were pooled and diluted with two volumes of Hz0, and the 0.20 ml was withdrawn and set aside. To the remaining 0.80 ml was product was isolated by lyophilization: 34.6 mg (59%); [aI2'D -11.5' (C 0.46, 5b% AcOH); TLC (A) 0.47, (B) 0.51, (C) 0.71, (D) added HBt (60 mg, 0.44 mmol). The solution was cooled to 0' and DCC (60 mg, 0.29 mmol) was added. After 1 hr at Oo and 1 hr at 0.73, (E) 0.79. Amino acid analysisz0 following 24-hr hydrolysis in room temperature the preactivation solution was added through a deaerated 6 N HCl at 110' gave the following ratios: Asp 1.01; Glu, fritted funnel to a solution of the peptide salt of 1 in DMF (1.2 ml) 2.88. 1.04; Pro, 1.03; Gly, 0.96; Ile, 1.00; Leu, 0.99; Dbt, 1.00; ":I, Following hydrolysis as above of a performic acid oxidized samwhich had been adjusted to pH 6.5 with i-PrzNEt (41 pl, 0.24 plezl the ratios were Cys(S03H), 1.08; Asp, 1.00; Glu, 1.03; Pro, mmol) along with 0.4 ml of DMF. The pH was maintained at 6-6.5 with additional aliquots of i-PrzNEt. After 36 hr the reaction had 0.96; Gly, 0.94; Ile, 0.96; Leu, 0.99; NHs, 2.96. Anal, stopped, with the ninhydrin test indicating approximately 30% of ( C ~ ~ H ~ ~ N I ~ O ~ Z SC, Z BH,~N. Z.~HZO) the free amino groups remaining. The remaining 0.20 ml of DMF Bioassays. Oxytocic assays were performed on isolated uteri solution of &Mpa(&Etz)(Ec) was preactivated with DCC in the from virgin rats in natural estrus according to the method of Holpresence of Hbt as described above and added to the reaction mixtonz2as modified by Munsickz3 with the use of Mg-free van DykeHasting's solution as bathing fluid. AVD assays were performed on ture. After 52 hr the product was precipitated by the addition of 95% EtOH (20 ml). The precipitate was isolated, washed by cenconscious chickens by the method of Coon,24 as described in the trifugation with EtOH, EtOAc,'and EtzO, and dried in vacuo: 162 US. as modified by Munsick, Sawyer, and van mg (50%).The filtrate was evaporated to dryness and the residue DykeJ6 Pressor assays were carried out on anesthetized male rats as described in the U S . P h a r m a c ~ p e i a . ~ ~ dissolved in boiling 95% EtOH (7 ml). The precipitate which ~D (c formed upon cooling was collected: 116 mg (36%);[ C X ] ~-43.5O 1.08, DMF). Both crops were identical by TLC (A, 0.79) and meltThe authors thank Ms. N i n a S m i t h S H, Z BN.~ ~ ' ~ H ZAcknowledgments. O) ing point (242' dec). Anal. ( C ~ ~ H S ~ N I ~ O ~ ~C, for the bioassays and Dr. Louis L. Nangeroni, New York [l-fl-Mercapto-fl.@-diethylpropionic acid,2-(3,5-dibromotyState Veterinary College at Cornel1 University, for the use rosine)]oxytocin. A solution of 3 (65.1 mg, 47.6 pmol) in liquid NH3 (freshly distilled from Na) was refluxed for 4 hr. The NH:] of his laboratory for the bioassay work. Particular apprewas removed by evaporation and lyophilization and the residue ciation is expressed t o Professor Vincent du Vigneaud for dissolved in H20 (50 ml) and MeOH (40 ml) under an Nz atmohis advice a n d s u p p o r t during the course of this investigasphere. A solution of ICHzCHzI (14.1 mg, 50 pmol) in 10 ml of tion. This work was supported in p a r t b y G r a n t HL-11680 MeOH was added and the disappearance of the sulfhydryl groups to Professor du Vigneaud from the National H e a r t a n d was followed by the Ellman method.Is The reaction was complete Lung Institute, U.S. Public Health Service. in 5 min, AcOH (5 ml) was added, and the solvents were removed the persistent blue color of excess Na was discharged with AcOH. The NH3 was removed by evaporation and lyophilization and the residue was dissolved in HzO (100 ml). The pH was adjusted to 1 with concentrated HC1 and the solution extracted with EtOAc. The EtOAc solution was dried with Na2S04 and evaporated to dryness. The colorless oil was dissolved in DMF (6 ml) and treated with freshly distilled ethyl isocyanate (0.35 ml, 4.4 mmol). After 48 hr the solution was evaporated (oil pump) to a colorless oil which had NMR (CDC13) proton ratios and chemical shifts consistent with expected values. The oil was dissolved in EtOAc (10 ml), and dicyclohexylamine (DCHA) (0.8 ml, 4 mmol) was added. The resulting crystalline precipitate was collected, washed with EtOAc, and dried in vacuo: 1.42 g (86%).The crude product was recrystallized from EtOH-EtOAc with 93% recovery: mp 128-129'. Anal. (CzzH4zNz03S) C, H, N.

1264 Journal of Medicinal Chemistry, 1975, Vol. 18, No. 12

References a n d Notes (1)J . Bid. Chem., 247,977 (1972). (2)D. B. Hope, V. V. S. Murti, and V. du Vigneaud, J . Biol. Chem., 237, 1563 (1962);B. M. Ferrier, D. Jarvis, and V. du Vigneaud, ibid., 240,4264 (1965). (3) H. Schulz and V. du Vigneaud, J . Med. Chem., 9,647 (1966); W. Y. Chan, H. Schulz, and V. du Vigneaud, IIIrd International Pharmacology Congress, Sao Paulo, Brazil, 1966, abstract 449;W. Y. Chan, R. Fear, and V. du Vigneaud, Endocrinology, 81,1267 (1967). (4)R. J. Vavrek, M. F. Ferger, G. A. Allen, D. H. Rich, A. T. Blomquist, and V. du Vigneaud, J . Med. Chem., 15, 123 (1972). (5) J. J. Nestor, Jr., M. F. Ferger, and V. du Vigneaud, J . Med. Chem., 18,284 (1975). (6)D.F. Dyckes, J. J. Nestor, Jr., M. F. Ferger, and V. du Vigneaud, J . Med. Chem., 17,250(1974). (7)H. D. Law and V. du Vigneaud, J. Am. Chem. Soc., 82,4579 (1960);K. Joit, J Rudinger, and F. Sorm, Collect. Czech. Chem. Commun., 26, 2496 (1961);28, 1706 (1963);Z.Berlnkovl, I. Rychlik, K. Joit, J. Rudinger, and F. Sorm, Collect. Czech. Chem. Commun., 26,2673 (1961);I. KrejEI, I. PollEek, and J. Rudinger, Br. J . Pharmacol. Chemother., 30, 506 (1967);A. L. Zhuze, K. JoHt, E. Kasafirek, and J. Rudinger, Collect. Czech. Chem. Commun., 29, 2648 (1964);J. Rudinger, Prog. Endocrinol., Proc. Znt. Congr. Endocrinol., 2nd, Part 11, 1202 (1965). (8)P. Marbach and J. Rudinger Experientia, 30,696 (1974).

Notes (9) E. 0.Lundell and M. F. Ferger, J . Med. Chern., 18, 1045 (1975). (10) St. Guttmann, Helv. Chim. Acta, 49,83 (1966). ( 1 1 ) E. 0.Lundell and M. F. Ferger, Bioorg. Chem., in press. (12) W. Konig and R. Geiger, Chem. Ber., 103,788 (1970). (13) F. Weygand and G. Zumach, 2. Naturforsch., B, 17, 807 (1962). (14) D. Yamashiro, Nature (London),201,76 (1964);D.Yamashiro, D. Gillessen, and V. du Vigneaud, J . Am. Chem. SOC.,88, 1310 (1966). (15) J. Porath and P. Flodin, Nature (London),183,1657 (1959). (16)E. Kaiser, R. L. Colescott, C. D. Bossinger, and P. I. Cook, Anal. Biochem., 34,595 (1970). (17) C. W. Smith, M. F. Ferger, and W. Y. Chan, J . Med. Chem., 18,822 (1975). (18) G. L.Ellman, Arch. Biochem. Biophys., 82,70(1959). (19) 0.H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193,265 (1951). (20)D. H. Spackman, W. H. Stein, and S. Moore, Anal. Chem., 30, 1190 (1958). (21) S.Moore, J. Bid. Chem., 238,235 (1963). (22)P. Holton, Br. J.Pharmacol. Chemother., 3,328 (1948). (23) R. A. Munsick, Endocrinology, 66,451 (1960). (24) J. M. Coon, Arch. Znt. Pharrnacodyn. Chemother., 62, 79 (1939). (25)“The Pharmacopeia of the United States of America”, 18th revision, Mack Publishing Co., Easton, Pa., 1970,p 469. (26) R. A. Munsick, W. H. Sawyer, and H. B. van Dyke, Endocrinology, 66,860(1960). (27)Reference 25, p 771.

3-Acyloxymethyl-7-(2-thienylacetamido)-3-cephem-4-carboxylic Acids. An Improved Synthesis and Biological Properties David A. Berges Research and Development Division, Smith Kline & French Laboratories, Philadelphia, Pennsylvania 19101. Received July 17, 1975 3-Acyloxymethyl-7-(2-thienylacetamido)-3-cephem-4-carboxylic acids have been prepared by an improved method utilizing the acylation of desacetylcephalothin with acid imidazolides. Their in vitro and in vivo antibacterial activities have been determined and compared with those of cephalothin.

Cephalothin (2, R = CHs), a commercially important cephalosporin, has good in vitro activity against a broad spectrum of gram-positive and gram-negative bacteria, but its in vivo activity is diminished by enzymatic hydrolysis of the acetate group to give the less active 3-hydroxymethyl compound 1.’ Testing the hypothesis that increasing the bulk of the R group in 2 would decrease susceptibility to enzymatic hydrolysis, Kukolja2 found only a small increase in in vivo activity relative to cephalothin for a series of cephalosporins in which R ranged from ethyl to cyclobutyl. Van Heyningen3 reported a significant drop in in vitro activity when R was an aromatic ring. Nevertheless, when R was 2-thienyl, the in vivo activity in mice was superior to that of cephalothin. With an interest in derivatives which possess in vitro activity equivalent to that of cephalothin but with greater metabolic stability, the variation of R (in particular, the introduction of heteroatoms) has been examined further. It has been reported that aroyl chlorides but not aliphatic acid chlorides acylate 7-acylaminodesacetylcephalosporanic acids.3 Aliphatic acid derivatives have been made by acylation of desacetylcephalosporanic acid esters with subsequent regeneration of the 4-carboxyl4 and by acylating 2-cephem acids followed by double bond isomerization.* I n contrast t o the acid chlorides, acylation of sodium 3hydroxymethyl-7-(2-thienylacetamido)-3-cephem-4-

carboxylate (1) with aliphatic acid imidazolides5 in an inert solvent (DMF) produced respectable yields6 of the 3-acyloxymethyl compounds 2. Lactonization and double bond migration, reported to be serious problems with the acid chloride acylations,’ were not observed with the acid imidazolides. (

CO,M 1

dOLM L

The new cephalosporins, which are summarized in Table I, were characterized by elemental analysis and ir and NMR spectroscopy.